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POM Exam 2 > Clinical Molecular Testing for Genetic Conditions > Flashcards

Clinical Molecular Testing for Genetic Conditions Flashcards

1
Q

primary types of molecular testing

A
  • genotyping (targeted variant testing)
  • sequencing of entire genes
  • deletion/duplication analysis of genes
  • genome-scale sequencing
  • DNA/protein analysis
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2
Q

silent mutations

A
  • results in codon with the same protein code
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3
Q

missense mutation

A
  • results in codon with different protein code
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4
Q

nonsense mutation

A
  • inserts stop codon
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5
Q

frameshift deltion

A
  • deletion/insertion that takes you out of frame
  • when you go out of frame, you typically run into a stop codon at some point
  • truncates message
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6
Q

robust PCR

A
  • only a small amount of DNA requires

- from a single cel you can amplify billions of copies of the region of interest

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7
Q

how PCR works

A
  • double stranded DNA is separated
  • forward and reverse primers anneal to homologous DNA
  • polymerase adds dNTPs
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8
Q

molecular genetic testing on cystic fibrosis

A
  • carrier testing
  • prenatal testing
  • neonatal screening
  • diagnosis
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9
Q

cystic fibrosis genetics

A
  • autosomal recessive disease
  • common among caucasians of Northern European ancestry
  • Ashkenazi Jews
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10
Q

cause of cystic fibrosis

A
  • mutation in CTFR gene

- over 1000 different mutations identified

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11
Q

cystic fibrosis results

A
  • defective chloride conductive results in thick mucus buildup In lungs,
  • GI tract
  • increase salt in sweat
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12
Q

ACOG/ACMG recommendations for cystic fibrosis testing

A
  • recommend mutation panel
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13
Q

mutation panel

A
  • includes 23 mutations in CTFR gene
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14
Q

when is the CTFR mutation panel recommended

A
  • CF carrier screening in women

- if positive, test partner

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15
Q

why not sequence entire CTFR gene?

A
  • cost/time

- interpretation of clinical significance of sequence variants

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16
Q

interpretation of clinical significance of sequence variants

A
  • novel sequence variants
  • variants of unknown significance
  • not all CTFR mutations are associated with classical cystic fibrosis
  • some mutations are associated with milder symptoms, atypical pancreatitis, or isolated CBAVD
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17
Q

hereditary breast and ovarian cancer syndrome genetics

A
  • autosomal dominant

- incomplete penetrance

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18
Q

hereditary breast and ovarian cancer syndrome risk

A
  • hereditary mutations in BRCA1 and BRCA 2 gene
  • risk of familial breast cancer (>50% risk)
  • risk of ovarian cancer (20-40% risk)
19
Q

incomplete penetrance

A
  • not everyone who has a deleterious variant manifests disease
20
Q

what reduces hereditary breast and ovarian cancer syndrome

A
  • prophylactic bilateral mastectomy (by 90%)

- oophorectomy (by 50%)

21
Q

hereditary break and ovarian cancer syndrome sequencing

A
  • need to sequence entire coding regions of these two genes to identify disease causing pathogenic mutations in presenting family member
  • subsequent testing may be targeted to a specific variant identified in the family
22
Q

Sanger/Dideoxy sequencing

A
  • PCR product is the template for the sequencing reaction
  • a fraction of dNTPs are fluorescently labelled ddNTPs, causing chain termination when incorporated
  • products are generated that are terminated at every nucleotide position
  • products are run on capillary gel electrophoresis instrument
  • software converts fluorescent signal to peaks for comparison to reference sequence
23
Q

benefits of Sanger sequencing

A
  • allows examination of DNA nucleotide sequence up to 500 bases
  • detects single base changes as well as insertions and deletions
24
Q

targeted analysis

A
  • mutations are known
  • few mutations are associated with disease
  • can test for individual mutations or sequence selected exons
  • faster, more economical
  • interpretation is more clear-cut
25
Q

gene sequencing

A
  • mutations are unknown
  • mutations occur throughout the gene
  • targeted mutation analysis is negative
  • more expensive
  • interpretation can be more complicated
26
Q

when to use PCR based methods

A
  • single nucleotide variants

- small insertions and deletions

27
Q

when to use deletion/duplication analysis

A
  • larger indels

- whole or partial gene deletions/duplications

28
Q

when to use cytogenetic karyotyping, FISH

A
  • genomic rearrangements
29
Q

clinical applications of massively parallel sequencing

A
  • genetic panels for specific conditions with multiple genes involved
  • whole exam sequencing
  • whole genome sequencing
30
Q

genetic panels for specific conditions with multiple genes involved

A
  • development delay
  • hearing loss
  • hereditary cancer syndromes
  • somatic mutations in tumors
31
Q

whole exome sequencing

A
  • sequencing of the coding region of all known human genes
32
Q

whole genome sequencing

A
  • sequencing of the entire human genome
33
Q

variants of uncertain significance

A
  • rare variants are common
  • just because we can detect a variant, doesn’t mean we know what it means
  • most variants have little to no clinical effect
34
Q

5-class scale for pathogenicity

A
  • benign
  • likely benign
  • variant of uncertain significance
  • likely pathogenic
  • pathogenic
35
Q

benign until proven otherwise

A
  • do not institute medical management
  • testing of family members not recommended
  • unless this would help with interpretation
36
Q

likely pathogenic/pathogenic

A
  • provided diagnosis and/or indication for medical management
  • targeted testing of family members can be performed
37
Q

what to consider when ordering molecular testing

A
  • why the test is being done
  • the best method for testing and limitations of available methods
  • discuss testing with geneticist or clinical lab director
38
Q

genetic linkage

A
  • violation of Mendel’s law of independent assortment when genetic loci are in close proximity
39
Q

linkage disequilibrium

A
  • refers to non-random association of alleles within a population
40
Q

clinical utility of linkage

A
  • use of segregation analysis to localize genes for condition
  • genetic testing for tightly-linked variants can be used when causative variant is not known
  • most variants identified by genome-wide association studies are not causative but are liked to causative alleles
41
Q

electrophoresis and blotting

A
  • applies a charge difference to induce movement through gel
  • move by size
    • smaller faster
  • proteins denatured to give more charge to mass ratio
  • blotting involves transfer from cell to more stable membrane, then use of targeted probe to visualize certain molecules
42
Q

southern blot

A
  • DNA probe to find DNA in sample
43
Q

northern blot

A
  • RNA probe to find RNA in sample
44
Q

western blot

A
  • antibody to find protein in sample

POM Exam 2 (21 decks)

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